The novel effect of Vernicia fordii extract on insulin secretion in pancreatic β-cells and improvement of. insulin resistance in diabetic mice. Therefore, restoring glucose-stimulated insulin secretion (GSIS) in the pancreas is effective therapy for type 2 diabetes. In pancreatic β-cells, VFE dramatically enhanced insulin release in a dose- and time-response manner and under hyperglycemia compared to euglycemia condition.
Orally administered VFE ameliorated hyperglycemia and improved insulin sensitivity in HFD-fed mice, and reduced pancreatic islet size compared with that observed in HFD-fed mice. This study establishes that VFE has a strong insulinotropic effect and improves insulin sensitivity by increasing glucose uptake and PKCα activation via intracellular Ca2+ influx. Keywords - Vernicia fordii, Glucose stimulated insulin secretion (GSIS), Intracellular calcium, Insulin sensitivity, Antidiabetic action.
MPLC fractions in VFE screening to confirm increased insulin release in MIN6 cells Figure 2-12.
Background
- Type 2 diabetes mellitus
- Glucose stimulated insulin secretion
- Natural products derived bioactive compounds treating diabetes
- Reference
LeRoith, D., Beta-cell dysfunction and insulin resistance in type 2 diabetes: the role of metabolic and genetic abnormalities. Wagner, Bridget K.; Altshuler, D., High-throughput luminescent reporter of insulin secretion for detecting regulators of pancreatic beta-cell function.
Vernicia fordii extract stimulates insulin secretion in pancreatic β-cells
Introduction
The effect of VFE on mitochondrial function was assessed from MIN6 cells in XFe24 microplates (Seahorse Bioscience, MA, USA). In addition, insulin release stimulated by TVFE was similar to that of 12-O-Tetradecanoylphorbol-13-acetate (PMA), which reportedly increases insulin secretion in pancreatic β-cells 13. Next, we fractionated TVFE, producing VFE as the most effective fraction in stimulating insulin secretion in MIN6 cells (Fig. 1.B.).
Since VFE stimulated insulin secretion in HG compared to LG, we hypothesized that VFE-induced insulin secretion is mediated by increased [Ca2+]i. In addition, we confirmed that VFE activated myristoylated alanine-rich C-kinase substrate (MARCKS), the main substrate of PKC that is involved in insulin secretion by actin rearrangement in pancreatic β cells (Fig. 3.A. and 3.B. ) 27. The specific PKC inhibitor Gö6983 significantly inhibited PKCα and MARCKS activation (Fig. 3.C.), and insulin secretion (Fig. 3.D.).
3.E., the insulinotropic effect of VFE was not altered by a specific PLC inhibitor, U73122 in MIN6 cells, indicating that VFE stimulates insulin secretion mainly through Ca2+-induced PKCα activation. These data support the notion that enhanced mitochondrial function including enhanced glucose uptake and ATP generation, is a vital mechanism for VFE-stimulated insulin secretion in MIN6 cells. Although neither VFE nor Glb altered body weight (Fig. 5.B.), the decrease in blood glucose induced by VFE was associated with an increased plasma insulin level after glucose administration (Fig. 5.C .).
These results indicate that the VFE glucose-lowering effect is the result of stimulated insulin secretion in pancreatic β-cells. The most active fractions 5-3 and 5-3-9 which were separated from VFE using prep-HPLC, were examined for the effect of insulin secretion in MIN6 cells to search for effective compounds (Fig. 11. and 14.). We showed that VFE regulates first- and second-phase insulin secretion in MIN6 cells in this study.
In addition, VFE-induced insulin secretion was enhanced by Ca2+-dependent activation of PKCα, which plays a major part in second-phase insulin release in pancreatic β-cells (Fig. 3.A. and 3.B .). We demonstrated that VFE potentiates glucose uptake in pancreatic β cells, implying that VFE modulates second-phase insulin secretion, which is a glucose-dependent process. Our results strongly suggest that VFE regulates mitochondrial function through increased glucose uptake and increases Ca2+ influx in MIN6 cells (Fig. 2.A. and 2.B.).
Furthermore, we demonstrate that inhibition of KATP due to the VFE-stimulated increase in ATP is an important mechanism for insulin secretion in MIN6 cells (Fig. 3.H. and 4.F.). Because PMA can mimic DAG to induce insulin secretion in pancreatic β-cells, we tested the effect of PLC on VFE-mediated insulin secretion. VFE-stimulated insulin secretion in proinsulin-Gaussia luciferase-transduced MIN6 cells in the absence or presence of [Ca2+]e under high glucose conditions.
Material and method
Result
Cell viability was not altered at concentrations <20 μg/ml of either chemical, indicating that TVFE and VFE potentiate insulin release without inducing cytotoxicity (Fig. 1.F.), thus these data suggest that VFE potentiates GSIS in pancreatic β cells. These results suggest that VFE in insulin release is primarily due to [Ca2+]e influx in pancreatic β cells. Increased [Ca2+]i has been shown to induce PKCα activation, which is a key mechanism for inducing GSIS in pancreatic β cells 13 , 26 .
Next, we investigated whether the disruption of [Ca2+]e influx or KATP activation regulates PKCα activation and insulin release in pancreatic β-cells. It has been well established that [Ca2+]e influx through either L-VDCCs or KATP is essential for insulin secretion 12. The inhibition of L-VDCCs with nifedipine, a specific L-VDCC blocker, suppressed VFE- induced PKCα activation and insulin secretion, indicating that [Ca2+]e influx via L-VDCCs at VFE increases insulin exocytosis (Figs. 3.F. and 3.G.).
Furthermore, the selective KATP activator diazoxide reduced PKCα activation and the insulinotropic effect of VFE (Figs. 3.H. and 3.I.). Glucose transport and metabolism in pancreatic β cells are essential processes associated with GSIS by regulating KATP 12 activity. Thus, we investigated the effect of VFE on GSIS by measuring intracellular glucose abundance and mitochondrial ATP production.
4.C. the activation of CREB by VFE was dose-dependent, indicating that VFE increases CREB activity and CREB-mediated gene expression to promote glucose uptake in pancreatic β-cells. Next, we estimated the effect of VFE on mitochondrial respiration using a seahorse cellular bioenergetic analyzer. Impaired insulin secretion is a critical pathophysiological feature of T2DM, and restoration of insulin secretion is an effective therapeutic option for diabetic complications 30 .
VFE dramatically alleviated glucose intolerance and insulin resistance of HFD-fed mice and improved fasting glucose levels in a dose-dependent manner (Fig. 6.D. and 6.E.). Decreased levels of HOMA-IR and increased levels of QUICKI clearly showed improvement in insulin sensitivity after treatment with VFE (Fig. 6.G. and 6.H.).
Discussion
Second, staurosporine, a potent PKC inhibitor, is reported to completely suppress PMA-induced insulin secretion 27 . However, it can also partially block GSIS, suggesting that staurosporine directly inhibits PKC, and alternative pathways may be involved in GSIS 38 . , besides [Ca2+]i, DAG produced by PLC is involved in the activation of PKCα.
In pancreatic β-cells, GSIS is regulated by several key genes, such as Glut2, Ins1 and PC. A glucose transporter deficiency has been reported in murine T2DM models and human T2DM patients, and its deficiency in the pancreas resulted in a loss of GSIS, leading to severe hyperglycemia 39. Previous results have shown that chronic high glucose exposure to β-cells is associated with blunted precursor insulin biosynthesis and lower Ins1 gene expression level 41 .
Pyruvate carboxylase (PC), a key enzyme of the tricarboxylic acid (TCA) cycle in mitochondria, is abundantly expressed in β-cells. In pancreatic β-cells, the PC pathway is believed to play an important role in pyruvate metabolism, insulin release, and cell proliferation 42. Similar to these results, we verified that VFE increased the expression of Glut2, Ins1, and PC together with glucose-dependent activation CREB (Figure 4C).
The upregulation of these glucose-regulated transcripts in cells by VFE treatment represents a remarkable glucose responsiveness of the β-cells, which could be a key aspect in the treatment of diabetes. Fuel‐dependent CREB activation is known to stimulate the expression of pancreatic β‐cell‐specific genes 29 , suggesting that VFE might promote the expression of those genes through glucose‐induced CREB activation. Because this was not determined in this study, further research is needed to determine the molecular mechanisms by which VFE regulates gene expression.
Conclusion
Figure
ΔF340/380 nm ratio at the consecutive time and the area under curve of ΔF340/380 nm ratio in each concentration of VFE treated MIN6 cells. -B) Western blot assay of pPKC substrates, PKCα and MARCKS phosphorylations followed by treatment of MIN6 cells with VFE in dose- (A) and time- (B) dependent manner. -D) Western blot assay (C) and insulin secretion (D) followed by treatment of MIN6 cells with VFE at the indicated concentrations alone or together with 10 μM Gö6983 (Gö).
-G) Western blot analysis (F) and insulin secretion (G) after treatment of MIN6 cells with VFE at the indicated concentrations alone or together with 10 μM nifedipine (Nif). -I) Western blot analysis (H) and insulin secretion (I) after treatment of MIN6 cells with VFE at the indicated concentrations alone or together with 200 μM diazoxide (Diaz). The relative ratio of pPKCα/PKCα or pMARCKS/MARCKS in all Western blot analyzes was quantified by ImageJ (AU; arbitrary unit).
The indicated concentrations of VFE and Glb were administered orally to mice before 30 minutes of 3 g/kg glucose oral injection. 30; the insulin measured in the blood collected before VFE or Glb injection, +30; the insulin measured in the blood collected after 30 minutes of glucose injection. -B) Body weight (A) and food intake (B) of HFD-fed mice treated with indicated concentrations of VFE and Glb or vehicle for 7 weeks.
OGTT and IPITT were performed in HFD-fed mice treated with vehicle, VFE and Glb. In western blot analysis, the relative ratio of pAkt/Akt was quantified by ImageJ (AU;. The relative fold change in the size of islets compared to NCD-fed mice was quantified by ImageJ.
Table
Ito, Y.; Yanase, S.; Tokuda, H.; Kishishita, M.; Ohigashi, H.; Hirota, M.; Koshimizu, K., Epstein-Barr virus activation by tung oil, extracts of Aleurites fordii and its diterpene ester 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate. Park, Y.-J.; Choe, Y.-h.; Kim, B.-S., Bovine viral diarrhea (BVD) virus antiviral activity of Korean Tung Tree (Aleurites fordii) extracts in vivo. J.; Jeong, H.; Baek, J.; Song, J.-H.; Kim, J.; Kang, H.; Han, S.-B.; Oh, S.-R.; Cho, S., Selective oncolytic effect in Epstein-Barr virus (EBV)-associated gastric carcinoma through efficient lytic induction by Euphorbia extracts.
Monauni, T.; Muggeo, M., Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity: studies in subjects with varying degrees of glucose tolerance and insulin sensitivity.
